DESCRIPTION: (Investigator's Abstract)A new approach to DNA sequencing is proposed, with potential applications both to large-scale genomic sequencing and to small-scale diagnostic sequencing. DNA templates with annealed primers will be tethered to agarose bead surfaces and interrogated serially, in the presence of exonuclease-free DNA polymerase, with separate flows of each of the four deoxynucleotide triphosphates (dNTP's), labeled with fluorescein at non-base-pairing sites. Because the polymerase enzyme is highly selective for the dNTP which is complementary to the template base at the primer extension site, the polymerase reaction to extend the primer will only occur when the correct dNTP is supplied; by sensing that extension has occurred and knowing the identity of the dNTP supplied, the sequence may be read. The fact that only a single type of dNTP is in contact with the primer/template duplexes at any one time ensures that extension of the primer strands proceeds synchronously so that the extension signals from all the strands reflect the base identity at exactly the same template sites. Extension will be sensed by detecting the fluorescence from the incorporated deoxynucleotides after excess unreacted dNTP's have been rinsed away. In order to allow continued detection of further extensions, the incorporated fluorescein labels will be photochemically destroyed after each readout. Sequence read lengths of several hundred bases are anticipated. Issues of polymerase fidelity and photochemical damage that can affect sequence accuracy and read length will be investigated, and the chemistry and the sequencing procedures will be adjusted to optimize, first, sequence read length, and subsequently, sequencing speed. A preliminary capillary flow device capable of semi-automated sequencing will be constructed. This novel new sequencing method may compete with conventional sequencing methods for genomic sequencing applications and in particular is aimed at allowing rapid and inexpensive diagnostic sequencing of targeted short regions of the human genome which code for proteins implicated in human disease.